During fertilization, there is a transient increase in the concentration of intracellular free calcium in the egg that is necessary for development to proceed. The molecular signaling pathway that links sperm-egg binding and fusion to this calcium increase is only now beginning to be understood. This study examines the regulation of the enzyme phosholipase C-gamma (PLC-g) during fertilization in the starfish. This enzyme is required for the fertilization calcium increase and embryonic development in the starfish. Recent work has shown that an src-like tyrosine kinase interacts with the starfish PLC-g enzyme at the time of fertilization. It is clear that several other tyrosine-phosphorylated molecules from the egg and sperm also interact with PLC-g at or during fertilization in the starfish, but the identity of these molecules is unknown. The objective of this proposed study is to examine the regulation of PLC-g during fertilization and to describe an interacting web of signaling proteins that function to stimulate egg activation during fertilization. This will be accomplished by 1) analyzing the localization of PLC-g in the live egg before, during and following fertilization with the use of photoactivatable green fluorescent (paGFP)-PLC fusion proteins; 2) identifying proteins that interact with PLC-g by affinity chromatography, followed by 2D PAGE and amino acid sequencing; 3) cloning of these molecules from a high-density arrayed starfish cDNA library, and the examination of the role these molecules play in fertilization by microinjection of dominant-negative fusion proteins and function-blocking antibodies. The information to be gained from these studies is pertinent to human health in a number of areas. Most directly, this will increase our knowledge about the fundamental mechanisms of fertilization, which could lead to advances in the study of infertility and contraception. Also valuable will be the knowledge gained about the regulation of signaling molecules, such as PLC-g and Src, which function in numerous cell types. [unreadable] [unreadable]